The invention relates to mononuclear cell expansion, and more particularly, to ex vivo expansion of hematopoietic stem cells (HSCs) derived from umbilical mononuclear cells.
All mature blood cells originate from a small population of hematopoietic stem cells (HSCs), which are characterized by their capacities to self-renew and the ability to differentiate into different hematopoietic cell lineages [Moore K A, et al. Blood. 1997; 89:4337-4347; McAdams T A, et al. Trends Biotechnol. 1996; 14:341-349]. The CD34 antigen, an integral membrane glycoprotein that functions as a regulator of hematopoietic cell adhesion to stromal cells within the marrow microenvironment, is expressed on human HSCs [Baum C M, et al. Proc Natl Acad Sci USA. 1992; 89:2804-2808; Guzman P F, et al. Arch Med Res. 2002; 33:107-114]. Cells that express high amount of CD34 antigen are described as early multipotential colony-forming unit (CFU)-Mix, CFU-blast and long-term culture-initiating cells (LTC-ICs), and their morphology appears as lymphocyte-like cells [Prosper F, et al. Blood. 1997; 89:3991-3997; Shih C C, et al. Blood. 1999; 94:1623-1636]. In clinical application, the number of CD34+ cells infused proved to be the major prognostic factor for engraftment and survival [Moore K A, et al. Blood. 1997; 89:4337-4347; McAdams T A, et al. Trends Biotechnol. 1996; 14:341-349; Shih C C, et al. Blood. 1999; 94:1623-1636]. Additionally, more and more studies have demonstrated that the CD34+CD38− fraction contains most clonogenic cells that can repopulate nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice [Danet G H, et al. Exp Hematol. 2001; 29:1465-1473; Zandstra P W, et al. Proc Natl Acad Sci USA. 1997; 94:4698-4703; Bhatia M, et al. Proc Natl Acad Sci USA. 1997; 94:5320-5325].
Umbilical cord blood (UCB), collected from the postpartum placenta and cord, has been identified as a rich source of HSCs, and provided as alternative to bone marrow transplantation [Gluckman E, et al. Bone Marrow Transplant. 1998; 22:68-74]. UCB transplantation has been used for treating hematopoietic disorders (leukemia, anemia, etc.), congenital immunodeficiencies, metabolic disorders, and autoimmune diseases [Rubinstein P, et al. N Engl J Med. 1998; 339:1565-1577; Kurtzberg J, et al. N Engl J Med. 1996; 335:157-166]. UCB transplantation in adults, however, has been limited by the concern that a single UCB unit does not contain sufficient number of CD34+ cells (the optimal dose for an adult is ≧2.5×106 CD34+ cells/kg) to rapidly reconstitute adult bone marrow function [Gilmore G L, et al. Exp Hematol. 2000; 28:1297-1305; McNiece I, et al. Exp Hematol. 2001; 29:3-11; McAdams T A, et al. Trends Biotechnol. 1996; 14:388-396]. Consequently, it is desirable if HSCs expansion ex vivo can be developed empirically without loss of their engraftment ability.
The dynamics of hematopoiesis are regulated by a delicate interplay of molecular signal and cellular microenvironment. Molecular signaling among cells is mainly achieved by means of secreted glycoproteins, also known as cytokines. One cytokine might have different effects on different types of cells, depending on the target cells, its concentration, and the presence of other cytokines [Guzman P F, et al. Arch Med Res. 2002; 33:107-114; Zandstra P W, et al. Proc Natl Acad Sci USA. 1997; 94:4698-4703; Gilmore G L, et al. Exp Hematol. 2000; 28:1297-1305; Yonemura Y, et al. Blood. 1997; 89:1915-1921; Lebkowski J S, et al. Stem Cells. 1995; 13:607-612; Audet J, et al. Biotechnol Bioeng. 2002; 80:393-404; Yao C L, et al. Enzyme Micro Technol. 2003; 33:343-352]. The cellular microenvironment, which is composed of stromal cells within bone marrow, is responsible for the fixation of HSCs by adhesion molecules, and also for the stromal cells secreted cytokines that promote HSC proliferation and differentiation [Baum C M, et al. Proc Natl Acad Sci USA. 1992; 89:2804-2808; Guzman P F, et al. Arch Med Res. 2002; 33:107-114; Prosper F, et al. Blood. 1997; 89:3991-3997; Shih C C, et al. Blood. 1999; 94:1623-1636; Yoo E S, et al. Stem Cells. 2003; 21:228-235; Rosler E, et al. Exp Hematol. 2000; 28:841-852]. However, the allogeneic or xenogeneic stromal cells in the co-culture system may induce immuno-responses when the ex vivo expanded HSCs are infused into patients. Serum, commonly used to support the culture of HSCs in many studies [Guzman P F, et al. Arch Med Res. 2002; 33:107-114; Prosper F, et al. Blood. 1997; 89:3991-3997; Shih C C, et al. Blood. 1999; 94:1623-1636; Gilmore G L, et al. Exp Hematol. 2000; 28:1297-1305; Yonemura Y, et al. Blood. 1997; 89:1915-1921], contains growth-required compounds including hormones, growth factors and binding proteins. However, serum is a potential source of bacterial, mycoplasmal and viral contaminations. Many reports have tried to develop serum-free media for ex vivo expansion of hematopoietic cells [Koller M R, et al. J Hematother. 1998; 7:413-423; Sandstrom C E, et al. Biotechnol Bioeng. 1994; 43:706-733; Mobest D, et al. Biotechnol Bioeng. 1998; 60:341-347; Bruyn C D, et al. Cytotherapy. 2003; 5:153-160]. To better conform to the clinical regulations, a stroma-free, serum-free, and chemically defined medium must be developed for HSCs expansion ex vivo.